Expression in Escherichia coli of the elongation factor 1beta gene and its nucleotide T160C mutant from the archaeon Sulfolobus solfataricus.
The guanine nucleotide exchange factor EF-1beta gene from the thermoacidophilic archaeon Sulfolobus solfataricus (SsEF-1beta) was amplified by PCR and cloned into the pT7-7 expression vector. One of four selected clones harbored the T160C nucleotide substitution leading to the Y54H amino acid change in a hydrophobic region of SsEF-1beta, caused by a nucleotide misincorporation of the Taq DNA polymerase during PCR. The resulting plasmids were used to transform the Escherichia coli BL21(DE3)pLysE strain. Upon induction with isopropyl beta-d-thiogalactopyranoside about 1.4 mg of the recombinant SsEF-1beta (recSsEF-1beta) and Y54HSsEF-1beta were obtained from 1 liter of cell culture. recSsEF-1beta and Y54HSsEF-1beta were both able to catalyze the GDP/GTP exchange on SsEF-1alpha as observed with the wild-type SsEF-1beta. In addition, the heat inactivation profiles of recSsEF-1beta and Y54HSsEF-1beta were identical, being both half inactivated after 30 min treatment at 105 degrees C. These results suggest that Tyr 54 is not essential for the nucleotide exchange activity and is not involved in the thermostability of SsEF-1beta.
Ianniciello
G
G
Dipartimento di Biochimica e Biotecnologie Mediche, Universitá di Napoli Federico II, via S. Pansini, 5, Napoli, I-80131, Italy.
Masullo
M
M
Raimo
G
G
Arcari
P
P
Bocchini
V
V
D019843:Archaeal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D019766:Genes, Archaeal; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D016944:Sulfolobus / Q000235:genetics* / Q000378:metabolism
D019843:Archaeal Proteins; D017931:DNA Primers; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate